Development of new methods for linking sugars to peptides or proteins is an active area of research (Brik A, Ficht S, Yang Y Y, Bennett C S and Wong C-H (2006) J Am Chem Soc 128: 15026-33; Brik A, Ficht S and Wong C-H (2006) Curr Opin Struct Biol 10: 638-44; Wu B, Chen J, Warren J D, Chen G, Hua Z and Danishefsky S J (2006) Angew Chem 45: 4116-45; Buskas J, Ingale S and Boons G J (2006) Glycobiology 16: 113R-136R.), because natural or neoglycoconjugates play important roles in biology and medicine and are indispensable tools for probing several biological processes (Doores K J, Gamblin D P and Davis B G (2006) Chem Eur J 12: 656-665).
However, despite dramatic progress in synthetic carbohydrate and protein chemistry in recent years, glycoconjugate synthesis involving sugar and a polypeptide remains a formidable task. This is principally because synthetic protocols are quite demanding and involve multiple reaction steps with requirements of rather extensive protection of reactive functionalities. The problem may be in part or completely obviated through the intermediary of enzymes. Indeed, glycosidases and glycosyl transferases, in appropriate situations, have made the oligosaccharide synthesis much simpler (Koeller K M and Wong C-H (2000) Chem Rev 100: 4465; Sears P and Wong C-H (2001) Science 291: 2344-2350.)
Given the current ease with which peptides are assembled by solid phase methodology and proteins obtained from expression systems, the availability of an enzyme capable of covalently linking a pre-synthesized sugar and a polypeptide would greatly facilitate the ‘convergent’ semisynthesis of glycopeptide or protein mimetics (neoglycoconjugates) with exquisite biological properties.
The bacterial transpeptidase sortase, present in the cell envelop of most gram-positive organisms, catalyzes the covalent anchoring of several virulence-related bacterial surface proteins to the peptidoglycan cross-bridges of the cell wall (Marraffini L A, Dedent A C and Schneewind O (2006) Microbiol Mol Biol Rev 70: 192-221). Sortase A of Staphylococcus aureus recognizes a LPXTG (SEQ ID NO: 11) like sequence motif located near the C-terminus of the target proteins, cleaves at Thr-Gly peptide bond, and catalyzes the formation of a new peptide bond between threonyl carboxyl and amino group of the peptidoglycan penta-glycine cross-bridges (Marraffini L A, Dedent A C and Schneewind O (2006) Microbiol Mol Biol Rev 70: 192-221; Ton-That H, Liu G, Mazmanian S K, Faull K F and Schneewind O (1999) Proc Natl Acad Sci USA 96: 12424-29). This is illustrated below:
—LPXTG (SEQ ID NO: 11)—+GGGGG (SEQ ID NO: 12)—→—LPXTGGGGG (SEQ ID NO: 13)—+G—
The transpeptidation reaction proceeds in two steps without the aid of ATP or any other extraneous molecule; active site cysteine residue first attacks the target LPXTG (SEQ ID NO: 11) substrate forming and acyl-enzyme intermediate which in the second step is resolved by the nucleophilic attack of the amino group of terminal Gly residue of the peptidoglycan. In the absence of a suitable amino nucleophile, LPXTG (SEQ ID NO: 11) peptide substrate is slowly hydrolyzed (Marraffini L A, Dedent A C and Schneewind O (2006) Microbiol Mol Biol Rev 70: 192-221; Ton-That H, Liu G, Mazmanian S K, Faull K F and Schneewind O (1999) Proc Natl Acad Sci USA 96: 12424-29). The ligation of LPXTG containing short or long polypeptide sequences to polypeptide fragments containing even a single Gly residue at the amino terminal has been shown to proceed in vitro (Mao H, Hart S A, Schink A and Pollok B A (2004) J Am Chem Soc 126: 2670-71). Mao et al (2004) have recently demonstrated the utility of sortase-mediated protein ligation as a tool for protein engineering by applying this approach to the synthesis of protein-peptide conjugates that would have been rather difficult to obtain by chemical or genetic methods.